The beneficial effects of estrogen on vascular physiology and disease involve two direct effects of estrogen on the blood vessel wall: rapid effects on vasomotor tone and longer-term inhibitory effects on vascular cell proliferation and atherosclerosis. Recent evidence supports that both of these involve estrogen-mediated increases in the bioavailibility and/or production of nitric oxide. In non-vascular cells, estrogen's effects are mediated through the classic estrogen receptor, now called ERa. Very recently, a second estrogen receptor, ERb, was discovered in rat prostate. Though ERa and ERb are homologous and share functional similarities, new data suggest differences in their tissue localization and transcriptional activities. This laboratory originally identified functional ERa in human vascular smooth muscle cells (VSMC) and functional ERa has been reported recently in vascular endothelial cells (EC) as well. They also reported that physiologic estrogen replacement markedly suppresses the response to vascular injury in ERa knockout (KO) mice to the same degree as in wild type animals, and that ERb is expressed in the vasculature of both normal and ERaKO mice. Preliminary data now show (I) ERb mRNA is expressed in both EC and VSMC; (ii) ERb mediates gene transactivation in EC and VSMC, including activation of inducible nitric oxide synthase (iNOS), and (iii) ERb, like ERa, can be activated in an estrogen-independent manner by growth factors. These findings support the central hypothesis of this application: the novel estrogen receptor, ERb, is an important mediator of the effects of estrogen on vascular cells and vascular injury. This hypothesis will be tested in the following specific aims: Specific Aim 1: Investigation of the distribution and relative level of expression of ERa and ERb mRNA and protein in normal and atherosclerotic murine and human vasculature; Specific Aim 2: investigation of the roles of estrogen receptors a and b in the mechanisms of endothelial (e)NOS and iNOS gene activation in EC and VSMC, respectively, and study of the molecular mechanisms of estrogen-independent activation of ERb; and Specific Aim 3: Study of the specific role of ERb in the inhibition of vascular injury by estrogen and in gene expression in eNOS KO and ERb KO mice. These studies will contribute to our understanding of the molecular mechanisms of estrogen's effects on vascular physiology and disease, and are thus directly relevant to the study of ischemic vascular diseases in both men and women.